Mononuclear phagocytes are closely associated with diseases of the central nervous system. Microglia found in normal adult brain are highly ramified, quiescent cells that retract processes and become reactive during CNS injury (Rio-Hortega, 1932). Reactive microglia (activated brain mononuclear phagocytes) have been identified with Alzheimer Disease (AD) neuritic plaques (Bolsi, 1927; McGeer et al., 1987; Rogers et al., 1988; Giulian, 1992; Perlmutter et al., 1992; Giulian et al., 1995a). As a result, xcex2 amyloid (Axcex2)-induced neuron damage is thought to involve inflammatory cells. In Alzheimer Disease, quantitative histopathology has determined that  greater than 80% of core plaques are associated with clusters of reactive microglia while fewer than 2% of diffuse Axcex2 deposits show such an association (Giulian et al., 1995a). These observations suggest that brain inflammatory responses may be directed specifically against the constituents of neuritic and core plaques. As the principal immune effector cells of the brain, activated microglia are capable of releasing such cytotoxic agents as proteolytic enzymes, cytokines, complement proteins, reactive oxygen intermediates, NMDA-like toxins, and nitric oxide (Thery et al., 1991; Giulian, 1992; Rogers et al., 1992; Lees, 1993, Banati, R. B., 1993).
Alzheimer Disease accounts for more than 15 million cases worldwide and is the most frequent cause of dementia in the elderly (Terry, R. D., Katzman, Bick, K. L. (eds), 1994) and is thought to involve mechanisms which destroy neurons and synaptic connections. The neuropathology of this disorder includes formation of senile plaques which contain aggregates of Axcex21-42 (Selkoe, D. J., 1991, Yankner and Mesulam, 1991; Price et al., 1992; Younkin, 1995). Senile plaques found within the gray matter of AD patients are in contact with reactive microglia and are associated with neuron damage (Terry, R. D., 1994 a and b, Masliah, E., et al., 1994, and Perlmutter, et al., 1992). Plaque components from microglial interactions with Axcex2 plaques tested in vitro were found to stimulate microglia to release a potent neurotoxic amine, thus linking reactive microgliosis with AD neuronal pathology (Giulian, et al., 1995). However, the plaque component, or components, which elicits neurotoxic responses in microglia remained elusive.
A second type of Axcex2 accumulation found in both AD and aged normal brain consists of diffuse plaques (discrete mesh-like structures of 70 to 100 xcexcm diameter, visualized by silver staining, thioflavine S, or immunohistochemistry) which are not associated with such pathological changes as dystrophic neurites or decline in cognitive function (Yamaguchi et al., 1988; Masliah et al., 1990, 1993). Finally, diffuse, amorphous deposits of Axcex2, demonstrable only by immunohistochemistry, have been described in aged brain and as an early manifestation of AD-like pathology in Down""s syndrome (Giaccone et al., 1989; Verga et al., 1989). Although the mechanisms which link neuritic and core plaques to dementia remain unresolved, two principal hypotheses have been advanced: 1) that AD acts as a potent and direct neurotoxic agent (Yankner et al., 1990) or 2) that neuritic/core plaques elicit a cascade of cellular events which lead to neuronal pathology (Davies, 1994; Giulian et al., 1995a). Support for the first hypothesis comes from in vitro observations in which synthetic Axcex2 peptides appear toxic to enriched cultures of neurons (Pike et al., 1991; Cotman et al., 1992) or to various non-neuronal cell lines (Behl et al., 1994; Pollack et al., 1995). Support for the second hypothesis comes from evidence that neuritic/core plaques are not directly neurotoxic, as shown by the fact that neurons can be grown successfully atop Axcex2 peptides (Koo et al., 1993; Wujek et al., 1996), that neuritic/core plaques added directly to neurons do not cause neuron damage (Giulian et al., 1995a), and that Axcex2 peptides infused into the brain do not cause tissue injury (Games et al. 1992; Podlisny et al., 1992; Stephenson and Clemens., 1992).
Since the description by Bolsi (1927) of reactive microglia near plaques in AD brain, it has been uncertain whether these reactive non-neuronal cells actually contribute to the disease process or merely reflect ongoing pathology. Recently, however, it has become clear that reactive microglia surround only certain types of amyloid deposits in the brain (the neuritic and core plaques) while ignoring nearby deposits of other types, including diffuse plaques (Perlmutter et al., 1992; Giulian et al., 1995a). Such selectivity in the distribution of reactive glia suggest that specific signals within neuritic and core plaques drive brain inflammation. With the increasing recognition that reactive microglia can mediate neuronal injury through release of cytotoxic factors (Banati et al., 1993; Giulian et al., 1993a), speculation on the involvement of microglia in AD has encompassed the release of complement proteins (Rogers et al., 1988, 1992), cytokines (Meda et al., 1995; Mrak et al., 1995), NMDA-like toxins (Piani et al., 1991; Giulian et al., 1995a, b), and free radicals (Thery et al., 1991; Hensley et al., 1994.
The present invention demonstrates that Axcex21-42 is the plaque-derived component which elicits neurotoxic responses in microglia. Importantly, the N-terminus of human Axcex2 provides an anchoring site necessary for initiating this neurotoxic cascade. More particularly, the HHQK-containing sequence is found to be significant in initiation. Because HHQK-like agents suppress toxic microgliosis in AD brain, neuronal loss and dementia may thereby be slowed. The present invention provides strategies for blocking the activation of microglia, thus making possible screening for therapies for the treatment of inflammatory injury to neurons in conditions such as Alzheimer Disease, stroke, trauma, multiple sclerosis (MS), Parkinson""s disease, HIV infection of the central nervous system, amyotrophic lateral sclerosis (ALS), hereditary hemorrhage with amyloidosis-Dutch type, cerebral amyloid angiopathy, cerebral amyloid angiopathy, Down""s syndrome, spongiform encephalopathy, Creutzfeld-Jakob disease, and the like.
The present invention is directed to a method of screening for an agent that inhibits the effect of a neurotoxin from a plaque component activated mononuclear phagocyte on a neuron comprising comparing measured mononuclear phagocyte-plaque component complex formation in the presence of an agent suspected of inhibiting complex formation to a measured control, wherein reduction of mononuclear phagocyte-plaque component complex formation compared to said control results in detection of an agent that inhibits mononuclear phagocyte-plaque component complex formation, and isolating a mononuclear phagocyte from an inhibited mononuclear phagocyte-plaque component complex formation, comparing said plaque component activation of a mononuclear phagocyte from an inhibited complex in the presence of an agent suspected of inhibiting activation to a measured control, wherein reduction of plaque component activation of said mononuclear phagocyte compared to said control results in detection of an agent that inhibits plaque component activation of mononuclear phagocytes and isolating a mononuclear phagocyte which is plaque component activation inhibited, comparing plaque component induced neurotoxicity of an activation inhibited mononuclear phagocyte in the presence of an agent suspected of inhibiting neurotoxicity to a measured control, wherein reduction of plaque component induced neurotoxicity compared to a control results in detection of an agent that inhibits a plaque component induced neurotoxic mononuclear phagocyte and isolating a plaque component induced neurotoxic mononuclear phagocyte, and measuring neuron function of a neuron in the presence of a neurotoxin from a plaque component activated mononuclear phagocyte or from a plaque component induced neurotoxic mononuclear phagocyte and an agent suspected of inhibiting the effect, wherein an increase in neuron function compared to a control results in detection of an agent that inhibits the effect of a neurotoxin from a plaque component activated mononuclear phagocyte on a neuron.
The present invention is directed to a method of screening for an agent that inhibits mononuclear phagocyte-plaque component complex formation by comparing mononuclear phagocyte-plaque component complex formation in the presence of an agent suspected of inhibiting the complex formation with a control such that a decrease in complex formation as compared to the control results in the detection of an agent that is inhibitory to mononuclear phagocyte-plaque component complex formation.
In addition, a method of screening for an agent that inhibits mononuclear phagocyte activation by a plaque component is embodied by the present invention and comprises comparing mononuclear phagocyte activation by a plaque component in the presence of an agent suspected of inhibiting the activation to a control such that a decrease in activation as compared to the control results in the detection of an agent that is inhibitory to mononuclear phagocyte activation by a plaque component.
Another method of the present invention is directed to screening for an agent that inhibits neurotoxicity of a mononuclear phagocyte which neurotoxicity is induced by a plaque component comprising comparing such neurotoxicity of a mononuclear phagocyte in the presence of an agent suspected of inhibiting neurotoxicity with a control where a decrease in mononuclear phagocyte neurotoxicity compared to the control results in the detection of an agent that is inhibitory to mononuclear phagocyte neurotoxicity induced by a plaque component.
A method of screening for an agent that inhibits the effect of a neurotoxin on a neuron, which neurotoxin is from a plaque component-activated mononuclear phagocyte comprising comparing neuron function of a neuron in the presence of a neurotoxin from a plaque component-activated mononuclear phagocyte and an agent suspected of inhibiting such effect to a measured control such that an increase in neuron function compared to said control results in detection of an agent that inhibits the effect of a neurotoxin from a plaque component activated a mononuclear phagocyte on neurons is yet another method embodied by the present invention.
Methods of measuring mononuclear phagocyte-plaque component complex formation, mononuclear phagocyte activation by a plaque component, mononuclear phagocyte neurotoxicity induced by a plaque component and the effect of a neurotoxin from a plaque component activated mononuclear phagocyte on a neuron include labeling one of the above-identified elements followed by imaging and amplifying nucleic acids from the mononuclear phagocyte or neuron and observing the amplified nucleic acids. Further to the forgoing detection methods, plaque suppressors may be measured by observing altered mononuclear phagocyte morphology, observing the expression of cell surface molecules on mononuclear phagocytes, and observing the release of nitric oxide, free radicals, cytokines, lipoproteins, enzymes, and proteins from mononuclear phagocytes. Furthermore, inactivators of neurotoxic mononuclear phagocytes may be detected by observing a loss of metabolic function, release of intracellular material, penetration of impermeant dyes, and reduction of cell number of neurons. Yet additional methods of detecting neurotoxic blockers include observing disruption of normal cell metabolism such as metabolism of glucose, the production of ATP, maintenance of ion gradients across a cell membrane, protein synthesis, nucleic acid synthesis, and mitochondrial respiration.
An agent is also provided by the present invention which agent is obtained by the process of screening for an agent suspected of inhibiting mononuclear phagocyte-plaque component complex formation, neurotoxicity of mononuclear phagocytes, and effects of neurotoxins on neurons. Agents having the sequence HHQK (SEQ ID NO: 1), chloroquine, tyramine, or an agent having activity similar to these agents, are objects of the present invention. A pharmaceutical compositions comprising an agent are also provided for by the present invention.